Process for preparing macrophages, kits, and composition for the use of this process

ABSTRACT

The invention relates to a process for preparing a composition comprising macrophages, optionally activated, and/or cells derived from monocytes with a strong potential for antigen presentation, said process comprising a stage of culture of monocytes present in the starting composition, this stage being preceding and/or followed by a stage of elimination of all or part of the constituents other than the monocytes present in the starting composition, with the aid of antibodies directed against said constituents, and/or followed by a stage of elutriation. The invention also concerns the compositions of kits for reducing this process to practice.

This is a continuation-in-part application of copending internationalapplication, PCT/FR96/00121 filed on Jan. 24, 1996, which designated theUnited States of America.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for preparing macrophages,specifically human macrophages, in particular activated macrophages(also designated as cytotoxic macrophages) or macrophages (or othercells derived from monocytes or from their precursors) for thepresentation of antigens, as well as kits and compositions which may beused for this process.

2. Description of the Related Art

Macrophages play a major role in the antitumoral response, and they areable to be activated by immunological activators against cancer cells(Adams D. and Hamilton T.: Activation of macrophages for tumor cellkill: effector mechanism and regulation. In Heppner & Fulton (eds),Macrophages and cancer. CRC Press, 1988, p. 27; Fidler I.: Macrophagesand metastases. A biological approach to cancer therapy. Cancer Res. 45:4714, 1985).

Furthermore, macrophages, or other cells derived from monocytes or fromtheir precursors, with their strong capacity for endocytosis, digestion,and surface antigen presentation, are capable of inducing a specificimmune response. In this way, they represent good candidates for thepreparation of vaccines, and more specifically cellular autologousvaccines. Macrophages, or other cells derived from monocytes, presentingantigens on their surface specifically capable of inducing a specificimmune response, are designated in the text which follows as MD-APCs(Monocyte-Derived Antigen Presenting Cells).

The MD-APCs are obtained by culture of mononucleated cells (monocytes ortheir precursors), taken from a patient or healthy individual, in thepresence of one (or several) antigen(s) which are desired to beexpressed as fragments on the surface of the cells obtained at the endof the culture period.

The antigens which may be cultured with the above mentionedmononucleated cells are, for example, antigens expressed by tumor cells(in particular fragments of killed tumor cells) or by pathogens (inparticular bacterial or viral protein fragments).

The MD-APCs thus obtained are used for the preparation of vaccinesdirected against the pathology associated with the antigen co-cultivatedwith the mononucleated cells (in particular vaccines against tumors orviral diseases) and more particularly for the preparation of autologouscellular vaccines which are advantageously administered to the patientfrom whom the starting mononucleated cells were taken.

MD-APCs are also able to be obtained by transfection of the abovementioned cells placed in culture, or of differentiated cells, inparticular macrophages, obtained after culture, with DNA sequencescoding for antigen fragments such as those defined above.

Macrophages presenting cytotoxic activity which is particularlysignificant (also designated as activated macrophages or MAK (registeredtrade mark)) as well as a culture medium and process for obtaining saidmacrophages were the subject matter of international patent applicationno. PCT/EP 93/01232, filed on May 18, 1993.

The process for preparing macrophages defined in the above mentionedinternational patent application is carried out with a composition richin blood cells obtained by apheresis from a healthy individual, andincludes a stage of monocyte culture in a culture medium containingvitamin D3 1.25-dihydroxyl and GM-CSF (Granulocyte-Macrophage colonystimulating factor). Advantageously, the monocytes are cultivated in thepresence of lymphocytes for about 6 to 7 days, and the differentiatedmacrophages thus obtained are activated in the culture medium by theaddition of interferon γ (IFN-γ).

This culture stage is preceded, as in all procedures for obtainingmacrophages described up to today, by a stage of separation of, in onepart, mononuclear cells, and, in another part, red cells, granulocytes,and part of the platelets contained in the composition derived fromblood obtained by apheresis; and by a stage of elimination, by washingpart of the blood platelets and anticoagulants remaining after thepreceding separation stage.

The above mentioned stage of separation of red cells and granulocytes isgenerally carried out by centrifugation of the medium containing themonocytes on a density gradient, particularly in a solution having aspecific weight of about 1.0 to about 1.3 g/ml, such as Ficoll Paquesolution (Pharmacia) with a specific weight of 1.077 g/ml.

In the above mentioned international patent application, the compositionderived from the blood is obtained by apheresis containing approximately7 to 9×10⁹ leukocytes in a volume of approximately 200 ml. After thestage of separation of the red cells and granulocytes by centrifugationon a density gradient, the composition of cellular suspension placed inculture thus contains, in a volume of approximately 500 ml to 1000 ml:

Hematocrit (Red cells)<0.1% of the number of the white cells present inthe composition

Platelets<10¹⁰

White cells 3 to 5×10⁹

Lymphocytes 50-70% of the number of white cells present in thecomposition

Monocytes 30-50% of the number of white cells present in the composition

Polynuclear cells (or granulocytes)<5% of the number of white cellspresent in the composition

The elimination of red cells and granulocytes, which is done beforehandin the culture stage, has always been considered as an indispensablestage, due particularly to the fact that the granulocytes are liable tototally or partially inhibit the differentiation of monocytes intomacrophages during the culture stage.

This inhibition would principally be due to the fact that red cells,granulocytes and platelets use the constituents of the culture mediumfor their own metabolism, thus exhausting the reserves necessary for thedevelopment of the monocytes, and leading to acidification of theculture medium in proportions such that the culture stage cannot becompleted (usually requiring about 6 to 7 days).

Furthermore, the granulocytes and platelets would be particularly liableto secrete suppresser factors, such as TGF (transforming growth factor),and prostaglandins, against the functionality of macrophages.

Therefore, it is currently acknowledged that the granulocytes and asmany of the platelets as possible should be separated from the monocytesbefore being placed in culture with the latter, even though thisseparation leads to the elimination of about 20% to about 50%, inparticular about 30%, of the mononucleated cells present in the mediumbefore separation.

SUMMARY OF THE INVENTION

The present invention aims at providing a new process for obtainingmacrophages or MD-APCs, optionally activated, which is the most simpleprocedure described up to today and presents the best results of all theprocesses for obtaining macrophages described up to today.

The invention also aims at providing kits (or cases) for carrying outthis process.

The invention also aims at providing for the user of said kits,compositions which can be used as internal controls in order to controlthe different stages of the process, particularly to control thecontents of monocytes, and optionally of other constituents of thestarting composition of the process of the invention, and to control thecontents of macrophages or MD-APCs, optionally activated, in the finalcomposition being sought.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated with the aid of the followingfigures:

FIG. 1 represents the assembly for the removal of platelets and washingthe product of apheresis,

FIG. 2 represents the assembly of the culture of mononucleated cells forobtaining macrophages or MD-APCs,

FIG. 3 represents the assembly of the elutriation system.

The present invention relates to a process for obtaining macrophages andMD-APCs, optionally activated, not including a stage of separation byphysical means, notably by centrifugation on a density gradient in theway described above, in one part, of the red cells and/or granulocytes,and, in another part, of other white cells, notably monocytes, presentin the composition derived from blood and obtained by apheresis.

The invention particularly relates to any process for preparing acomposition containing macrophages or MD-APCs, optionally activated,characterized in that, starting from a composition derived from blood ofhuman origin, rich in blood cells and specifically in white cells suchas monocytes, in particular a composition derived from blood such asthat obtained by apheresis, said procedure includes the followingstages:

advantageously a dilution of said composition derived from blood,particularly in approximately 2 to 3 times the volume of the latter,with the aid of an appropriate physiologic solution,

a stage of washing said composition derived from blood, advantageouslyby simple centrifugation, and washing the pellet issued from the saidcentrifugation, after recovery of the pellet in an appropriatephysiologic washing solution, particularly in a bag (a transfer typebag), effected, for example, by pressure on said bag, thus eliminatingthe washing solution to another bag or other recipient, to recover acomposition depleted of possible anticoagulants, various residues andplatelets,

optionally, the repetition of the washing stage mentioned above,particularly from 1 to 2 times,

a culture stage of the cells contained in the composition derived fromblood obtained after the washing stage mentioned above, by placing saidcells in an appropriate culture medium, in particular in a bag,advantageously hydrophobic, for about 6 to 7 days. This culture stage isoptionally done in the presence of specific antigens mentioned above,that is, in particular, in the presence of antigens characteristic oftumor cells or of pathogens in the case of MD-APC production, andpossibly followed by an activation stage of the macrophages or MD-APCsobtained in the culture medium, by addition in said culture medium of anactivator such as interferon γ, said activator being placed in contactwith the contents of the culture medium for about 16 to 24 hours. Thisstage of culture, and optionally activation, are possibly followed bycentrifugation, in particular following the procedure indicated above,this stage of culture, and optionally activation, being:

preceded by a stage of elimination of all or part of the constituentsother than the monocytes or their precursors, in particular theplatelets, red cells, granulocytes, and lymphocytes, liable to bepresent in the starting composition, by contacting the compositionderived from the blood obtained after the stage of washing preceding thestage of culture, with antibodies directed against all or part of theabove mentioned constituents, and recovering the solution containing themonocytes or their precursors, all or part of the above mentionedconstituents remaining fixed to the antibodies;

and/or followed by a stage of elimination of all or part of theconstituents other than the macrophages or MD-APCs by being placed incontact with the composition derived from blood obtained after theculture stage with the antibodies such as described above, and recoveryof the solution containing the macrophages or MD-APCs , all or part ofthe above mentioned constituents remaining fixed to the antibodies,

and/or followed by a purification stage, in particular an elutriation,in which the macrophages or MD-APCs, optionally activated, are separatedfrom the other constituents of the composition obtained after the stageof culture, and possibly the stage of activation mentioned above,separated from, in particular, the platelets, red cells and lymphocytes,by physical means.

The invention results from the discovery made by the inventors whichshows that, contrary to ideas known in the field, it is possible tobring the culture of monocytes or their precursors into macrophages orMD-APCS to completion with good results when the culture medium containscontrolled quantities of red cells, lymphocytes, granulocytes, andplatelets.

The invention thus offers the advantage of having access to a processfor obtaining macrophages or MD-APCs which enables to get rid of thestage of separation by physical means of, in one part, the mononucleatedcells, and more specifically the monocytes, and, in another part, thered cells, granulocytes (and the vast majority if not the totality ofplatelets) contained in the composition derived from the blood obtainedby apheresis, said stage being costly (requiring sophisticated material)and time consuming.

Advantageously, in the above mentioned process of the invention, thestarting composition derived from the blood of human origin, namely thecomposition of cellular suspension enriched in blood cells, such as thatissued from apheresis and before the stage of washing preceding thestage of culture in the procedure described above, is such that itincludes a proportion of monocytes greater than about 5%, in particularabout 10% to 30%, in number of white cells present in said composition.

Such a starting composition is advantageously obtained by the reductionto practice of process of apheresis conducted on a patient, this processbeing in particular carried out according to any technique and with anymaterial known by any individual skilled in the art, optionallyaccording to a process adapted for obtaining a starting composition suchas defined here above and hereafter.

Advantageously, the starting composition derived from blood of humanorigin mentioned above can contain up to about 3% red cells, andpreferably is such that it comprises a proportion of red cells less thanabout 1%, in particular from 0.3% to 0.5%, in number of white cellspresent in said composition.

Advantageously, the original composition derived from blood of humanorigin mentioned above is such that:

the number of platelets is between about 2×10¹¹ to about 6×10¹¹ in avolume of about 150 ml to about 200 ml,

the number of white cells is between about 5×10⁹ to about 5×10¹⁰ in avolume of about 150 to about 200 ml,

the proportion of lymphocytes is between about 60% to about 80%, innumber of white cells present in the said composition,

the proportion of monocytes is between about 10% to about 30%, in numberof white cells present in said composition,

the proportion of granulocytes is between about 10% to about 20%, innumber of white cells present in said composition.

The stage of washing the starting composition, preferably repeated 2times, permits the essential elimination of a part of the platelets, andoptionally of the reagents used during the process of obtaining theoriginal composition, in particular those of apheresis, such asanticoagulants. Advantageously, the percentage of platelets eliminatedduring this stage is about 80% to about 90%.

The composition obtained after the stage of washing the originalcomposition contains advantageously, in a volume of about 600 ml, thefollowing constituents:

Hematocrit: 0.2% to 0.4% in number of white cells present in saidcomposition

Platelets: about 2 to about 6×10¹⁰

White cells: about 4×10⁹ to about 4×10¹⁰

Lymphocytes: about 60 to about 80% in number of white cells present insaid composition

Monocytes: about 10% to about 20% in number of white cells present insaid composition

Polynuclear cells: about 10 to about 15% in number of white cellspresent in said composition.

According to a preferred embodiment of the process of the invention, thestage of culture and differentiation of the monocytes or theirprecursors into macrophages or MD-APCs is carried out from a compositionderived from blood of human origin containing mononucleated cells,namely monocytes or their precursors and lymphocytes, as well as redcells (in appropriate quantities, in particular in the proportionsindicated above), granulocytes and platelets, said culture stage beingcarried out in particular starting from the composition obtained afterthe stage of washing the starting composition, such as is describedabove.

Concerning the stage of culture itself, it preferably takes place over aperiod of about 6 to about 8 days in a specific ready to use culturemedium based on modified IMDM (Iscove Modified Dubelcco Medium, Gibco),namely the Dubelcco medium modified by Iscove (IMDM) to which is addedL-Glutamine (2 mM, Gibco) or advantageously L-Alanyl-L-Glutamine. (2 mM,Gibco), pyruvic acid (2 mM, Gibco), Indomethacine (5×10⁻⁶ M, Sigma),mercaptoethanol (3×10⁻⁵ M, Gibco), and non-essential amino acids (2%,Gibco). At the time of culture, the medium will be supplemented by 2 to5% AB⁺ serum (or by autologous serum) as well as with antibiotics suchas penicillin and streptomycin.

The invention more specifically relates to the above mentioned ready touse culture medium, containing L-Alanyl-L-Glutamine instead ofL-Glutamine. The inventors have in fact shown evidence that the abovementioned ready to use culture medium containing L-Alanyl-L-Glutaminecan advantageously be conserved at 4° C. with the group of constituentsof the kits of the invention described hereafter, while the abovementioned ready to use culture medium containing L-Glutamine must beconserved at -20° C.

For the differentiation of monocytes into macrophages, the abovementioned culture medium is advantageously supplemented by GMCSF andvitamin D3.

For the differentiation of monocytes or their precursors into MD-APCs,the above mentioned culture medium is used for co-culture with theantigens described above, and is advantageously supplemented by one ormany cytokines such as GMCSF, TNF-α, IL-13, IL-4, and/or by otheradjuvants such as calcitriol and histamine.

In the case of obtaining activated macrophages or MD-APCs, an activator,such as interferon γ, is advantageously introduced in the abovementioned culture medium about 16 to about 24 hours before the end ofthe culture.

According to a preferred embodiment of the procedure described above,the composition of the cellular suspension obtained after the culturestage, in a volume of about 600 ml to 2000 ml, is advantageously thefollowing:

Lymphocytes: about 2×10⁹ to about 2×10¹⁰

Macrophages (or MD-APCs): about 6×10⁸ to about 6×10⁹

This final composition includes little or no granulocytes, these havingfor the most part decomposed during the stage of culture.

The yields of the culture stage mentioned above are advantageously about80% mononucleated cells, and about 50% to about 70% for thedifferentiation into macrophages or MD-APCs (taking into account thenumber of starting monocytes).

The above mentioned yields are calculated as the ratio between thenumber of macrophages or MD-APCs obtained after the culture stage andthe number of monocytes present in the composition before culture.

Advantageously, in the preferred embodiment of the process of theinvention described above, the above mentioned stage of culture, andoptionally activation, is followed by a stage of purification, inparticular by elutriation, in which the macrophages or MD-APCs,optionally activated, are separated from the other constituents of thecomposition obtained after the stage of culture, and optional activationmentioned above, separated in particular from the platelets, red cellsand lymphocytes, by physical means.

Advantageously, the stage of culture, and optional activation, carriedout in the preferred process mentioned above, is followed by a stage ofelutriation represented in FIG. 3, this being preferably carried outusing the following mechanism:

a bag (designated B4 in FIG. 3) containing the composition comprisingmacrophages or MD-APCs, optionally activated, obtained after the stageof culture, and after optional activation, as well as a bag containing aphysiologic solution (also designated as elutriation solution) able tocontain the entire elutriation system in a medium viable for themacrophages or MD-APCs, optionally activated, and an air intake arelinked to a transfuser with three branches equipped with a filter,

once the air intake is closed, a peristaltic pump pulls the contents ofthe bag B4 into an elutriator equipped with a rotor with an elutriationchamber, the feeding of the entirety of the elutriation circuit withphysiologic liquid being assured by the pulling of the contents of thebag containing the elutriation solution, with the above mentioned pump,during the entire duration of the elutriation,

the speed of rotation of the elutriation rotor and the delivery from thepump are such that the various constituents of the composition of bagB4, entering the rotor, are separated as a function of their size in theelutriation chamber; the constituents of the smallest size migratefirstly to the top of the chamber (near the exit of the elutriator),while the constituents of the largest size, namely the macrophages orMD-APCs, optionally activated, migrate to the exit of the chamberlastly,

the different constituents of the composition in bag B4 thus separatedare harvested separately into bags linked to the elutriator; theplatelets and various residues of small size are recovered first intobag C1; the red cells are then recovered into bag C2, the lymphocytesinto bags C3 and C4, and finally the macrophages or MD-APCs into bag E1,the different categories of constituents mentioned above being extractedfrom the elutriator as a function of their respective size, either bydiminishing the speed of rotation of the elutriator, or by augmentingthe delivery of the pump. The recovery of macrophages or MD-APCs intobag E1 is advantageously able to be carried out by stopping the rotor ofthe elutriator and opening the air intake; bag E1 thus contains thecomposition of macrophages or MD-APCs, optionally activated,advantageously in an essentially pure form.

As a variation of the preferred process described above, the culturestage and differentiation of monocytes or their precursors intomacrophages or MD-APCs may be preceded by a stage of elimination of allor part of the constituents other than the mononucleated cells,specifically those other than the monocytes liable to be contained inthe composition obtained after the stage of washing the originalcomposition, such as is described above, said elimination stage beingnot carried out by physical means, but by carrying out immunologicalreactions between all or part of the above mentioned constituents andantibodies able to recognize said constituents.

The stage of elimination by antibodies mentioned above is advantageouslycarried out by contacting the composition issued from the stage ofwashing the starting composition with anti-platelet antibodies, and/oranti-red cell antibodies, and/or anti-granulocyte antibodies, fixed on asolid support, in particular on the walls of a transfer type bag, or onthe walls of any other device in which said composition issued from theinitial stage of washing is able to be introduced, said device beingoptionally set up in order to allow the circulation of said compositionalong the walls on which said antibodies are fixed.

Contacting said composition with said antibodies is carried out eithersimultaneously with all or part of the antibodies mentioned above, orsuccessively in particular by introduction of said composition into abag or other device such as is described above containing anti-plateletantibodies, then passing said composition into a second bag containinganti-red cell antibodies, and/or passing said composition into a thirdbag containing anti-granulocyte antibodies, the lymphocytes beingeliminated during the stage of elutriation following the stage ofculture.

According to another preferred embodiment of the process of theinvention which specifically does not include a stage of elutriation,the included stage of culture, and optional activation, is:

preceded by a stage of contacting the composition issued from the stageof washing the starting composition with the anti-platelet antibodies,and/or anti-red cell antibodies and/or anti-granulocyte antibodies,fixed on a solid support, in particular in the manner indicated above,

contacting said composition with said antibodies being carried out,either simultaneously with all or part of the above mentionedantibodies, or successively in particular in the manner indicated above,

said composition being then transferred into a bag containing theculture medium,

and followed by a stage of contacting the composition issued from saidstage of culture, and optional activation, with the anti-lymphocyteantibodies, by introduction of said composition into one (or several)bag(s) or other device such as described above, containinganti-lymphocyte antibodies, and recovery the composition of macrophagesor MD-APCs, optionally activated, being sought, in an essentially pureform.

Advantageously, when the preferred process above described is carriedout by contacting the composition issued from the stage of washing thestarting composition in successive contact with anti-red cellantibodies, then anti-platelet antibodies, then anti-granulocyteantibodies, the composition of the cellular suspension thus obtainedbefore the stage of culture, in a volume of about 600 ml to about 2000ml, is advantageously the following:

Red cells:<0.1%

Platelets:<10¹⁰

White cells: about 3×10⁹ to about 3×10¹⁰

Lymphocytes: about 60% to about 80% in number of white cells present insaid composition

Monocytes: about 20% to about 30% in number of white cells present insaid composition

Polynuclear cells:<5%.

In the preferred process described above, the composition of thecellular suspension obtained after the culture stage, in a volume ofabout 600 ml to 2000 ml, is advantageously identical to that of thecellular suspension obtained after the culture stage in the precedingpreferred procedure, not requiring a stage of immunological reactionprevious to the culture stage, namely,

Lymphocytes: about 2×10⁹ to about 2×10¹⁰

Macrophages: about 6×10⁸ to about 6×10⁹

The yields of the culture stage mentioned above are advantageouslyidentical to those described in the preceding preferred procedure,namely about 80% mononucleated cells, and about 50% to about 70% for thedifferentiation into macrophages (taking into account the startingnumber of monocytes).

The yields of the processes described above are advantageously of thesame order, be these processes carried out according to the principle ofelutriation after the culture stage, or according to the principle ofthe immunological reaction before and after the culture stage.

Thus, the above described processes of the invention are such that onecan obtain cellular suspensions comprising macrophages or MD-APCs,optionally activated, in an essentially pure form, in particular inproportions of about 80% to about 95%, and with final yields of about20% to about 50% (said yields being calculated as a ratio between thequantity of purified macrophages or MD-APCs obtained at the end and thequantity of monocytes initially placed in culture).

According to a specifically advantageous embodiment of the processes ofthe invention described above, the composition of the starting cellularsuspension, and/or at least one of the compositions of cellularsuspension obtained under the different stages of the above mentionedprocedures, and/or the composition of the final cellular suspension,is/are able to be compared to the compositions called internal controls,in order to make a good follow up of the processes of the invention.

An internal control particularly advantageous is that which isconstituted of a determined quantity of monocytes, purified from bloodtaken from a healthy individual, advantageously fixed, in particular bylyophilization, said internal control allowing for the quantification ofthe number of monocytes present in the starting composition.

An internal control specifically preferred is that which includes about2×10⁶ to about 4×10⁶ lyophilized monocytes in a volume of about 1 ml toabout 2 ml of an aqueous solution.

The quantification of the number of monocytes present in the originalcomposition is advantageously carried out by withdrawing an aliquot partof the starting composition, labelling the monocytes liable to bepresent in the fraction withdrawn, in particular with the aid oflabelled antibodies, for example by radioactivity, enzymes, or byfluoresceine, revealing the labelling carried out by using anyappropriate device, and comparing the intensity of the labellingobtained under the same operating conditions with said internal control.

The labelled antibodies able to recognize specific surface antigens ofmonocytes are advantageously those directed against, for example, thefollowing antigens: CD14, HLADR, and CD64.

Another internal control specifically advantageous is that constitutedfrom a determined quantity of macrophages or purified MD-APCs,optionally activated, from blood withdrawn from a healthy individual,advantageously fixed, in particular by lyophilization, thenreconstituted in an adequate solution of 1 to 2 ml, said internalcontrol allowing for the quantification of the number of macrophages orMD-APCs, optionally activated, present in the final composition.

An internal control specifically preferred is such that it includesabout 2×10⁶ to about 4×10⁶ lyophilized macrophages in a volume of about1 ml to about 2 ml of an aqueous solution.

The quantification of the number of macrophages present in the originalcomposition is advantageously carried out by withdrawing an aliquot partof the original composition, labelling the monocytes present in thefraction withdrawn, in particular with the aid of labelled antibodies,for example by radioactivity, enzymes, or by fluoresceine, revealing theeffected labelling by using any appropriate device, and comparing theintensity of the labelling obtained under the same operating conditionswith said internal control.

The labelled antibodies able to recognize specific surface antigens ofmacrophages and/or MD-APCs are advantageously directed against at leastone of the following antigens: CD54, CD58, CD80, CD86, CD14, CD16,MAX-1, CD64, HLADR described in particular in the following articles:Andreesen et al., Biology 47: 490-497 (1990), Lopez et al., Journal ofImmunotherapy 11: 209-217 (1992), Chokri et al., Anticancer Research 12:2257-2260 (1992).

Advantageously, the internal controls mentioned above are constitutedfrom beads, chosen in particular from among fluorescent beads, having adiameter comparable to that of monocytes or macrophages, and able togive signals identical to those given by monocytes and macrophages in,respectively, the starting and final solutions.

According to another specifically advantageous embodiment of theprocesses of the invention described above, the control of thecompositions of macrophages or MD-APCs, optionally activated, obtainedwhen reducing to practice these processes, can be carried out bydetection, and optionally measure of cytokines secreted by themacrophages in said compositions, and compared to the standard secretionprofile of cytokines by macrophages or MD-APCs, optionally activated.

As an illustration, the detection of secreted cytokines such as IL-1(Interleukin 1) and TNF-α (Tumor Necrosis Factor-α) can be carried outby withdrawing an aliquot part of culture medium in which thedifferentiation and the activation of macrophages obtained has takenplace.

The macrophages and M-APCs, optionally activated, obtained by theprocess of the invention, may be tested for their capacity to recognizeand kill tumor cells (in particular by cytotoxicity test) or, in thecase of MD-APCs, for their capacity for antigenic presentation, inparticular by verifying their capacity to induce allogeniclymphoproliferation (for example the MLR test, Mixed LymphocyteReaction). These tests allow to determine the number of functional unitsof macrophages or MD-APCs obtained during the process of the invention.

The invention also relates to kits (or cases) for obtaining compositionsof macrophages or MD-APCs, optionally activated, characterized in thatthey comprise:

the necessary elements for washing, culture, and elutriation, including:

one or several washing bag(s),

one or several culture bag(s), preferably hydrophobic,

one or several connector(s) and needle(s) for the elimination of thesupernatant of the washings,

one or several connection tube(s) between the preceding bags,

optionally, one or several injection site(s) in the washing and culturebag(s),

one or several systems allowing for the opening or the closing of thelinks between the washing bag(s) and the culture bag(s), in particular,systems of clamps on the tubes,

two bags containing the physiologic solution for the washing and theelutriation,

a three branch transfuser with air intake,

a silicon tube and a joint on the rotor serving as a connection betweenthe transfuser and the rotor of the elutriator,

one (or several) collection bag(s), optionally linked together by tubesequipped with a clamp system,

a tube and joint on the rotor serving as a connection between thecollection bag(s) mentioned above and the rotor of the elutriator,

the reactive agents necessary for the culture, differentiation,activation, and control, including:

a bag of specific, ready to use culture medium, in particular atprimarily constituted from modified IMDM, namely Iscove modifiedDubelcco medium (IMDM, Gibco), to which has been added L-Glutamine (2mM, Gibco) or L-Alanyl-L-Glutamine (2 mM, Gibco), pyruvic acid (2 mM,Gibco), Indomethacine (5×10⁻⁶ M, Sigma), mercaptoethanol (3×10⁻⁵ M,Gibco), nonessential amino acids (2%, Gibco), and 2 to 5% of AB⁺ serum(or autologous serum), as well as antibiotics such as penicillin andstreptomycin, added at the moment of culture,

one or several supplements advantageously under liquid or lyophilizedform, to be added to the culture medium, chosen from among thefollowing:

GMSCF,

vitamin D3,

interleukin-13 (IL-13)

IL-4

TNF-α

histamine

optionally, an activator such as interferon γ, advantageously underliquid or lyophilized form,

advantageously one (or several) internal control(s) comprisingmonocytes, and/or macrophages or MD-APCs, the latter being optionallyactivated, advantageously fixed, or lyophilized, and optionallylabelled, or calibrated beads, optionally labelled, in particular byfluorescence,

one or several labelled antibodies, in particular by fluorescence, suchas those directed against antigens CD3 marker for lymphocytes used incase of negative control), CD45 marker for leukocytes in general), CD14,CD16, CD64, CD54, CD58, CD80, CD86, HLA-DR and MAX-1.

A kit specifically advantageous in the framework of the presentinvention is presented in the form of two groups:

group I contains the bags, tubes, injection sites, clamp systems, andthree branch transfuser, namely, the "dry" elements used for washing,culture, and the purification by elutriation,

group II contains a bag containing the specific culture medium as wellas the reactive agents for the culture, differentiation, activation, andquality control.

The kit mentioned above is moreover characterized in that:

group I is constituted of two sub-groups in the form of two boxes:

box A containing the material necessary for washing the cells obtainedfrom apheresis, in particular:

three bags with a capacity of 600 ml (central bag A1, and recovery bagsA2 and A3 shown in FIG. 1),

a transfer set for transferring the washing solution into the bag (bagA1 in FIG. 1) in which is transferred the blood composition in order todilute and wash the cells issued from apheresis,

a transfer set for the washed cells (contained in bag A1 in FIG. 1) tothe culture bags (bags B1, B2, and B3 in FIG. 2),

a transfer set for the culture medium, firstly towards the washed cells(contained in bag A1 in FIG. 1), and secondly towards the culture bagsmentioned above,

three hydrophobic culture bags with a capacity of 3 liters (bags B1, B2,and B3 in FIG. 2),

box B contains the elements for the separation of the differentiatedmacrophages or MD-APCs from the other cells present in the cultureduring the stage of elutriation, in particular:

a gathering bag (or pooling bag B4 in FIG. 2),

a three branch transfuser with air intake,

a silicon tube, two joints to the rotor, the tube and one of the twojoints serving as a connection between the transfuser and the rotor ofthe elutriator, the other joint serving as a connection with said rotorand the group of bags mentioned hereafter, the latter tube containing asite for sample withdrawal,

a combination of four recovery bags (bags C1, C2, C3, and C4 in FIG. 3)with a capacity of one liter, a bag (bag E1 in FIG. 3) with a capacityof 600 ml to contain the final fraction of purified macrophages orMD-APCs, and a bag (bag E2 in FIG. 3) with a capacity of 600 ml for therecovery of the supernatant of the preceding bag, and a bag (bag E3 inFIG. 3) for injecting the patients with macrophages or MD-APCs,optionally activated,

group II is represented by box C, an isothermal box, optionally with acooling system to maintain the temperature at 4° C., and containing inparticular:

a bag of 2 liters of culture medium primarily constituted from modifiedIMDM, as described above,

a box containing the following liquid or lyophilized products:

a flask of antibiotics (penicillin, advantageously 1,000 U/streptomycin,advantageously 1,000 μg),

a flask of vitamin D3 (advantageously 4 to 8 μg),

a flask of GMSCF (advantageously 5×10⁵ to 10⁶ U),

a flask of interferon γ (advantageously 25×10⁴ to 5×10⁵ U),

a flask of IL-13,

a flask of IL-4,

a flask of TNFα,

a flask of histamine,

a box containing one (or several) internal control(s) as describedabove, and one or many flasks containing the labelled antibodiesanti-CD3, anti-CD45, anti-CD14, anti-CD16, anti-CD64, anti-MAX-1,anti-CD54, anti-CD58, anti-CD80, anti-CD86, and anti-HLA-DR, inparticular labelled with fluoresceine.

The invention also refers to the use of a kit as defined above for therealization of a process as described above according to the inventionincluding a stage of elutriation.

The invention also relates to a kit for obtaining compositions ofmacrophages or MD-APCs, optionally activated, characterized in that itincludes:

one (or several) bag(s) inside of which fixed anti-red cell antibodiesare found,

one (or several) bag(s) inside of which fixed anti-platelet antibodiesare found,

one (or several) bag(s) inside of which fixed anti-granulocyteantibodies are found,

one (or several) bag(s) inside of which fixed anti-lymphocyte antibodiesare found,

one (or several) culture bag(s),

one (or several) bag(s) for the recovery of purified macrophages orMD-APCs, optionally activated,

linking tubes between the different bags mentioned above,

supplements advantageously under liquid or lyophilized form, to be addedto the culture medium, in particular:

antibiotics such as penicillin and streptomycin,

GMCSF,

vitamin D3,

IL-13

IL-4

TNFα

histamine

optionally, an activator such as interferon γ, advantageously underliquid or lyophilized form,

advantageously one (or several) internal control(s) comprisingmonocytes, or macrophages or MD-APCs, optionally activated,advantageously fixed, and optionally labelled, or of calibrated beads,optionally labelled, in particular by fluorescence, such as is describedabove,

labelled antibodies, in particular by fluorescence, such as thosedirected against the antigens CD3, CD45, CD14, CD16, CD64, CD54, CD58,CD80, CD86, MAX-1 and HLA-DR.

The invention relates more specifically to the use of a kit as describedabove for carrying out a process as described above according to theinvention comprising a stage of immunological reaction.

The invention also concerns any liquid composition containing a cellularsuspension derived from blood of human origin, characterized in that itincludes a proportion of monocytes greater than about 5%, in particularabout 10% to about 30%, in number of white cells present in saidcomposition.

The invention also relates to any composition such as described above,characterized in that it includes a proportion of red cells less thanabout 3% or less than about 1%, in particular from 0.3% to 0.5%, innumber of while cells present in said composition.

The invention relates more specifically to any composition such asdescribed above, characterized in that:

the number of platelets is between about 2×10¹¹ to about 6×10¹¹ in avolume of about 150 ml to about 200 ml,

the number of white cells is between about 5×10⁹ to about 5×10¹⁰ in avolume of about 150 ml to about 200 ml,

the proportion of lymphocytes is between about 60% to about 80%, innumber of white cells present in the said composition,

the proportion of granulocytes is between about 10% to about 20% innumber of white cells present in said composition.

The invention refers more specifically to the above mentionedcompositions such as obtained in the process of apheresis, carried outin particular on a volume of blood of approximately 5 to 10 liters.

The invention also relates to the use of a composition such as describedabove for obtaining a composition of macrophages or MD-APCs, optionallyactivated, in particular by carrying out a process according to theinvention, said composition being able to be administered to a patient,optionally after dilution of the macrophages or MD-APCs, optionallyactivated, thus obtained in an appropriate injectable solution.

The invention also relates to the compositions (in particularpharmaceuticals or vaccines) containing macrophages and/or MD-APCs,optionally activated, in particularly in the proportions of purityindicated above, and such as are obtained by carrying out the process ofthe invention, optionally in association with an acceptablepharmaceutical vehicle.

The invention also relates to the compositions which can be used asinternal controls in the framework of reducing to practice a processaccording to the invention, such as described above.

As an example, the internal control in the framework of the preparationof activated macrophages is presented in the following manner:

Contents

1 flask of lyophilisate (3×10⁶ /flask)

1 flask of reconstitution solution.

Reconstitution

Rehydrate the flask of lyophilisate with 1 ml of the solution ofreconstitution solution.

Mix gently by turning. Wait at least 10 minutes before using thepreparation.

Use

the volume of internal control used is 100 μl (3×10⁵ cells)

add the antibody to be tested

incubate for 30 minutes at room temperature

add 1 ml of PSB (do not wash)

analyze by cytofluorometry

The percentage and intensity of labelling of freshly prepared MAK orMD-APCs will be compared to the cells produced.

Table 1 which follows gives an indication of the evolution of membranemarkers in relation to time of storage, of the lyophilized macrophagesused as internal controls. The results are expressed as percentage ofpositive cells and intensity of labelling. The line "Ctrl" correspondsto the results obtained with fresh, non-lyophilized macrophages,cultivated and obtained according to the procedure of the invention. Theline "7 Days" corresponds to macrophages obtained according to theprocess of the invention, lyophilized and rehydrated 7 months afterlyophilization. An overall statement can be made that the percentages ofpositive cells as well as the intensities of labelling are keptequivalent to or similar to the fresh macrophages and those which areconserved in a lyophilized state for 7 days or 7 months. Thus, the userof the kits of the invention is able to compare the compositions whichwill be obtained with the internal controls (or standards) furnishedwith the kit, said controls being advantageously produced according tothe same process and being lyophilized.

                  TABLE 1                                                         ______________________________________                                        Markers                                                                       CD45        CD3     CD14    CD16  CD64  HLADR                                 ______________________________________                                        Ctrl    92/115  0.3/54  87/149                                                                              22/35 77/63 84/115                              7 Days  95/131  3/30    96/119                                                                              10/30 93/59 96/169                              7 Months                                                                              91/119  3/22    86/95 10/25 81/54 82/116                              ______________________________________                                    

Advantageously, the kits of the invention are accompanied by writtenprocedure forms for the preparation of macrophages or MD-APCs,optionally activated.

Advantageously, the kits of the invention are also accompanied byautomated computerized instructions to guide the user through theprocesses and validate each step of the processes of the invention, thefinal goal being to determine the above mentioned number of functionalunits obtained by carrying out the processes of the invention.

As an illustration, the written procedure forms for the step ofactivating macrophages, accompanying a kit according to the invention,can be presented in the following manner: ##STR1##

Description of the Figures

The description of the operations represented by FIGS. 1 through 3 is asfollows:

I--REMOVAL OF PLATELET FROM THE PRODUCT OF APHERESIS (FIG. 1)

(1) Transfer the contents of the apheresis bag into bag A1 (600 mlcapacity).

(2) To the bag containing the mononucleated cells, add a weight ofbuffered saline solution such that the final weight of the bag is450-500 g.

(3) Centrifuge bag A1 at 300 g for 10 minutes at +4° C.

(4) Transfer the supernatant into bag A2, using a press.

(5) Fill to 450 g with saline solution.

(6) Centrifuge bag A1 a second time.

(7) Transfer the supernatant into bag A3.

Detach the cells from the centrifugation bag A1 by friction.

(8) Fill to 600 ml with culture medium which has been previouslyprepared.

Note the volume of culture medium added.

Withdraw the control samples under sterile conditions.

II--CULTURE OF MONONUCLEATED CELLS (FIG. 2)

(9) Distribute equally into the culture bags B1, B2 and B3.

(10) Fill the volume required for each bag with culture medium.

(11) At the end of the culture and activation periods, transfer thecontents of the three culture bags into the pooling bag B4.

III--SEPARATION BY ELUTRIATION (FIG. 3)

Attach part I (entry of Kit) to the entry of the sterilized elutriationchamber and the exit of the kit (part II) to the exit of the elutriationchamber

(12) Fill the circuit with at least 300 ml of elutriation solution.

(13) Simultaneously, close the entryway of the elutriation solution andopen that of the cellular suspension bag (bag B4) without changing thedelivery of the pump.

As soon as all of the cells have passed, close the entryway of the bagB4 of cells and open that of the elutriation solution.

Increase the delivery of the pump every 500 ml and withdraw a sample.

(14) When cells having the size of macrophages begin to exit, stop thecentrifugation and deviated the withdrawal to the collection bag E1 byclosing the entryway of the elutriation solution and opening the airintake.

(15) Centrifuge bag E1 at 400 g for 10 min.

(16) Transfer the supernatant into bag E2.

(17) Put the cells back into suspension in a solution of 250 ml of humanalbumin at 4%. Homogenize well.

(18) Transfer into the injection bag E3.

We claim:
 1. A process for preparing a composition comprising at leastone of macrophages, activated macrophages, cells derived from monocytes,precursors of the cells derived from monocytes presenting specificsurface antigens which are capable of inducing a specific immuneresponse, said MD-APC's being optionally MD-APCs, and activated MD-APCs,said composition being prepared from a composition derived from blood ofhuman origin, comprising the steps of:diluting said composition derivedfrom blood with an appropriate physiologic solution, washing saiddiluted composition by simple centrifugation, and washing a pelletissued from said centrifugation, after recovery of the pellet, in aappropriate physiologic washing solution in a transfer bag, by exertingpressure on said transfer bag, the washing solution thus beingeliminated to a recipient container, to recover a washed compositiondeprived of possible anticoagulants and various residues andimpoverished in platelets, optionally, repeating the washing step,culturing cells contained in the washed composition by placing it in anappropriate culture medium in a bag for about 6 to about 10 days, saidculturing optionally taking place in the presence of antigens,optionally, activating at least one of macrophages and MD-APCS obtainedin the culture medium, by adding an activator in said culture medium,said activator being placed in contact with contents of the culturemedium for about 16 to about 24 hours, optionally, performing a secondcentrifugation of the culture medium and washing a pellet issued fromthe second centrifugation,wherein said process further comprises atleast one of the following steps: prior to the culturing and optionalactivation step, eliminating at least part of constituents other thanthe monocytes or their precursors, able to be present in the startingcomposition by contacting the washed composition with antibodiesdirected against at least part of the constituents, and recovering asolution containing at least one of monocytes and their precursors, atleast part of the constituents remaining fixed to the antibodies,following the culturing and optional activation step, eliminating atleast part of constituents other than the macrophages or MD-APCs byplacing the composition derived from blood obtained after the culturestep in contact with antibodies directed against at least part of theconstituents other than the macrophages or the MD-APCs, and recovering asolution containing the macrophages or MD-APCs, at least part of theconstituents other than the macrophages remaining fixed to theantibodies, following the culturing and optional activation step,purifying by separating the macrophages of MD-APC from the otherconstituents of the composition obtained after the culturing andoptional activation step by physical means.
 2. A process according toclaim 1, wherein the starting composition derived from blood of humanorigin includes a proportion of monocytes greater than about 5% innumber of white cells present in said composition.
 3. A processaccording to claim 1, wherein the starting composition derived fromblood of human origin includes a proportion of red cells less than about3% in number of white cells present in said composition.
 4. A processaccording to claim 1, wherein the starting composition derived fromblood of human origin is such that:a number of platelets is betweenabout 2×10¹¹ to about 6×10¹¹ in about 150 ml to about 200 ml, a numberof white cells is between about 5×10⁹ to about 5×10¹⁰ in about 150 ml toabout 200 ml, a proportion of lymphocytes is between about 60% to about80%, in number of white cells present in the said composition, aproportion of granulocytes is between about 10% to about 20% in numberof white cells present in the said composition.
 5. A process accordingto claim 1, wherein the steps of culturing, activation andcentrifugation are followed by a stage of elutriation carried out byusing the following mechanism:an elutriation bag containing thecomposition comprising activated macrophages or MD-APCs, optionallyactivated, obtained after the step of culturing, and optionalactivation, as well as a bag containing a physiologic solution able tocontain the entire elutriation system in a medium viable for themacrophages or MD-APCs, optionally activated, and an air intake, arelinked to a transfuser with three branches equipped with a filter, oncethe air intake is closed, a peristaltic pump pulls the contents of theelutriation bag into an elutriator equipped with a rotor with anelutriation chamber, the feeding of the entirety of the elutriationcircuit with physiologic liquid being assured by pulling of the contentsof the bag containing the elutriation solution, with the above mentionedpump, during the entire duration of the elutriation, the speed ofrotation of the elutriation rotor and the delivery from the pump aresuch that the various constituents of the composition of the elutriationbag, entering the rotor, are separated as a function of their size intothe elutriation chamber, the constituents of the smallest size migratefirst to the top of the chamber, while the constituents of the largestsize, namely the macrophages or MD-APCs, optionally activated, migrateto the exit of the chamber last, the different constituents of thecomposition in the elutriation bag thus separated are harvestedseparately into bags linked to the elutriator, the platelets and varioussmall size residues being recovered first into bag C1, the red cells,the lymphocytes the macrophages being recovered into a plurality ofrecovery bags, the different categories of constituents mentioned abovethus being extracted from the elutriator as a function of theirrespective size, by one of diminishing the speed of the rotation of theelutriator and increasing the delivery of the pump, the recovery ofmacrophages or MD-APCs into a macrophage/MD-APC bag advantageously beingable to be carried out by stopping the rotor of the elutriator andopening the air intake, the macrophage/MD-APC bag thus containing thecomposition of macrophages or MD-APCs, optionally activated, beingsought, in an essentially pure form.
 6. A process according to claim 1,wherein the step of culturing, and optional activation, is:preceded by astep of contacting the washed composition with at least one ofanti-platelet and anti-red cell and anti-granulocyte antibodies fixed ona solid support, recovering a resulting solution which is thentransferred into a bag containing the culture medium, and followed by astep of contacting the composition issued from the stage of culturing,and optional activation, with anti-lymphocyte antibodies, byintroduction of said composition into at least one container containinganti-lymphocyte antibodies, and recovery of the composition being soughtof macrophages or MD-APCs, optionally activated, in an essentially pureform.
 7. The process of claim 1, wherein the volume of the physiologicsolution used in the dilution step is approximately 2 to 3 times thevolume of the composition derived from blood with which the physiologicsolution is mixed.
 8. The process of claim 1, wherein the optionalrepeated washing step is performed twice.
 9. The process of claim 1,wherein the bag used for the culturing step is hydrophobic.
 10. Theprocess of claim 1, wherein the washed composition is placed in theculture medium for about 6 to 7 days.
 11. The process of claim 1,wherein the antigens optionally used in the culturing step are antigenscharacteristic of at least one of tumor cells and pathogens.
 12. Theprocess of claim 1, wherein the activator in the culture medium isinterferon γ.
 13. The process of claim 1, wherein the constituents otherthan the monocytes or their precursors optionally eliminated areplatelets, red cells, granulocytes, and lymphocytes.
 14. The process ofclaim 1, wherein the optional purification step is performed byelutriation.
 15. The process of claim 14, wherein the other constituentsfrom which the macrophages or MD-APC are separated in the optionalpurification step are platelets, red cells, and lymphocytes.
 16. Theprocess of claim 2, wherein the proportion of monocytes to white cellsis in the range of about 10% to about 30%.
 17. The process of claim 3,wherein the proportion of red cells to white cells present in thecomposition is in the range of 0.3% to 0.5%.
 18. The process of claim 1,wherein:the volume of the physiologic solution used in the dilution stepis approximately 2 to 3 times the volume of the composition derived fromblood with which the physiologic solution is mixed; the optionalrepeated washing step is performed twice; the bag used for the culturingstep is hydrophobic; and the washed composition is placed in the culturemedium for about 6 to 7 days.
 19. The process of claim 18, wherein theantigens optionally used in the culturing step are antigenscharacteristic of at least one of tumor cells and pathogens, and theactivator in the culture medium is interferon γ.
 20. The process ofclaim 19, wherein:the constituents other than the monocytes or theirprecursors optionally eliminated are platelets, red cells, granulocytes,and lymphocytes; the optional purification step is performed byelutriation; and the other constituents from which the macrophages orMD-APC are separated in the optional purification step are platelets,red cells, and lymphocytes.